Ultrasound contrast agents (UCA) are microbubbles with a gas core stabilized by a shell, that act as echo-enhancers to improve sensitivity and specificity in ultrasonic diagnosis. Poly (lactic acid) (PLA) UCA have been previously developed in our laboratory. In the previous studies, PLA UCA have been shown to have a high acoustic enhancement both in vitro and in vivo. Combining US with other imaging modalities such as fluorescence, magnetic resonance (MRI) or computerized tomography (CT), can improve the accuracy of many ultrasound (US) applications and provide more comprehensive imaging information. In this study, we functionalized our current UCA by encapsulating aqueous and organic quantum dots (QD) for fluorescence imaging, magnetic iron oxide nanoparticles (MNP) for MRI, or gold nanoparticles (Au-NP) for computed tomography (CT) contrast (Fig. 1).UCA were prepared with a water-in-oil-in-water-emulsion technique. Incorporation of the QD, MNP or Au-NP was accomplished by adding the solids to the first emulsion phase to encapsulate the agents into the shell of the UCA. In vitro acoustic testing was performed on a custom-built acoustic setup. The 5 MHz transducer was focused in a 37oC water bath through a sample holder containing phosphate buffered saline (PBS) and connected to a pulser/receiver (Panametrics Waltham, MA) to generate an acoustic pulse. The reflected signal was detected with the transducer then displayed on an oscilloscope (Lecroy 9350 A Chestnut Ridge, NY). Labview7 Express (National Instruments, Austin, TX) was used for data acquisition and processing. Environmental scanning electron microscopy (SEM) (FEI XL30, Hillsboro, OR) was used to observe the surface morphology of loaded UCA. Transmission electron microscopy (TEM) (JEM 1010, JEOL) was performed at an accelerating voltage of 80 kV to investigate the microstructure of the agent. Confocal microscopy was performed using an Olympus IX81microscope run by Olympus Fluorview version 1.7b (Olympus Corporation, Tokyo) to visualize the florescent properties of the QD loaded UCA. The MRI relaxation times of MNP loaded UCA were determined with relaxometry (60 MHz, Minispec, Bruker). Phantom imaging of MNP-UCA was done using a 3T clinical system. The CT attenuation rate and phantom imaging of Au-NP loaded UCA was determined using a clinical scanner. Biocompatibility was assessed using the LIVE/DEAD assay and HepG2 and RAW 264.7 cells.SEM images show that the loaded UCA formed smooth spheres. TEM images show that MNP are distributed uniformly on the shells of the UCA, whereas gold particles are aggregated (Fig. 2). Confocal microscopy indicates that the fluorescent properties of both types of encapsulated QD are retained (Fig. 3). The loaded UCA show decreases in acoustic enhancement (~1dB for both QD loaded UCA and ~4 dB for MNP and Au-NP loaded UCA) compared with the unloaded controls (18.62 dB), however, this reduced value should still provide adequate signal for clinical use (Fig. 4). CT phantom scanning of Au-NP loaded UCA show the CT attenuation rate as 5.8 HU/mM. The longitudinal, r1 and transverse, r2 relaxivities were found to be 1.667 and 118 mM1s1 and a r2/r1 ratio of 70.77. Phantom MR imaging supported this result, where strong signal loss can be seen when imaging of MNP-UCA was done using T2-weighted sequences (Fig. 5). Cell viability assays indicated that the agents were biocompatible even at doses 50 time greater than those expected in vivo.The current results show that QD, MNP and Au-NP can be encapsulated within the shell of the UCA and retain their individual contrast properties without affecting ultrasound contrast. These labeled microbubbles could provide an improved platform technology for dual imaging approaches.